1. Field of the Invention
This invention relates to a signal processing method for determining base sequence of nucleic acids.
2. Description of the Prior Art
It is essential to obtain genetic information carried by organisms in order to make the function or replicated mechanism of the organism clear in the field of molecular biology which has been rapidly developed in recent years. Particularly, it is essential to determine base sequence of nucleic acids such as DNA (or DNA fragment; the same applies hereinbelow) which carries specific genetic information.
Maxam-Gilbert method and Sanger-Coulson method are known as typical methods for determining the base sequence of nucleic acids such as DNA and RNA. In the former Maxam-Gilbert method, a group containing a radioactive isotope such as .sup.32 P is attached to a chain molecule of DNA or a DNA fragment at one end to label it with the radioactive element and then the bond between the constitutional units of the chain molecular is base-specifically cleaved by a chemical reaction. A mixture of the resulting base-specific DNA cleavage products is resolved through gel electrophoresis to obtain a resolved pattern (not visible) wherein each of the numerous cleavage products is resolved on the gel support medium. The resolved pattern is visualized on a radiographic film such as an X-ray film to obtain an autoradiograph thereof as a visible image. The bases in certain positional relationships with the end of the radioactive element-attached chain molecular can be sequentially determined according to the visualized autoradiograph and the applied base-specific cleavage means. In this way, the sequence for all bases of the DNA specimen can be determined.
In the latter Sanger-Coulson method, synthetic DNA products which are complementary to the chain molecule of DNA or DNA fragment and radioactively labeled, are basespecifically synthesized by utilizing a chemical reaction, and the obtained mixture of numerous synthetic DNA products is resolved on a support medium by gel electrophoresis to obtain a resolved pattern. In a similar manner to that described above, the base sequence of DNA can be determined according to the visualized autoradiograph.
For the purpose of carrying out the determination of the base sequence of nucleic acids simply with high accuracy in autoradiography, there are described in U.S. application Ser. No. 549,417, abandoned (now pending as U.S. application Ser. No. 837,037) and U.S. application Ser. No. 902,101, a continuation of abandoned U.S. application No. 664,405 autoradiographic procedures which utilize a radiation image recording and reproducing method using a stimulable phosphor sheet, in place of conventional radiography using a radiosensitive material such as an X-ray film. The stimulable phosphor sheet comprises a stimulable phosphor and has such properties that when exposed to a radiation, the stimulable phosphor absorbs a portion of radiation energy and then emits light (stimulated emission) corresponding to the radiation energy stored therein upon excitation with an electromagnetic wave (stimulating rays) such as visible light or infrared rays. According to this method, exposure time can be greatly shortened and there is no fear of causing problems such as chemical fog associated with prior arts. Further, since the autoradiograph having information on radioactively labeled substances is stored in the phosphor sheet as radiation energy and then read out as stimulated emission in time sequence, information can be expressed by the form of numerals and/or symbols in addition to image.
The base sequence of the nucleic acids has been conventionally determined by visually judging individual resolved positions of the base-specific cleavage products or the base-specific synthetic products of radioactively labeled nucleic acid (hereinafter referred to simply base-specific fragments of nucleic acid) on the autoradiograph and comparing them among the resolved rows thereof. Namely, the analysis of the autoradiogrpah is done by observing the visualized autoradiograph with eyes, and such visual analysis requires great amounts of time and labor.
Further, since the visual analysis of the autoradiograph varies or fluctuates owing to the skill of investigators, the results on the determination of the base sequence of nucleic acid vary depending on the investigators and the accuracy of information is limited to a certain extent.
In order to improve the accuracy of the information, there are proposed in pending U.S. application Nos. 024,909 continued from 865,956 and 568,877, both now abandoned, and U.S. application Ser. No. 730,034 methods for automatically determining the base sequence of DNA by obtaining the autoradiograph as digital signals and subjecting the digital signals to appropriate signal processing. The digital signals corresponding to the autoradiograph of the radioactively labeled substances can be obtained either by visualizing the autoradiograph on a radiographic film and photoelectrically reading out the visible image on said film by means of reflected light or transmitted light when the conventional radiography is employed, or by directly reading out the stimulable phosphor sheet without the visualization of the autoradiograph when the radiation image recording and reproducing method is employed.
However, the resolved pattern obtained by resolving (developing) radioactively labeled substances on a support medium by electrophoresis or the like is liable to cause various distortion and noise. A typical example thereof is offset distortion, that is, the deviation (slippage) of the position of the overall rows from one another due to differing resolution starting position or starting time among the rows. The offset distortion is caused mainly by that the shapes (the size of recess) of many slots (sample introducing ports) provided in the upper part of a support medium such as a gel medium are not always identical and there is a difference between individuals. There also bring about the offset distortion that positions on which the sample is to be deposited are deviated from one another in introducing the sample into the support medium and that the penetration rates of the sample into the support medium are different from one another when the washing of urea out of the gel medium is insufficient just before the introduction of the sample.
FIG. 1 shows an example where a resolved pattern causes offset distortion due to unevenness in the shapes of slots. FIG. 1-(a) shows the upper part of a support medium which has been used for the resolution of a sample and FIG. 1-(b) shows a resolved pattern obtained by introducing the same sample into each of slots (1) to (4) and then resolving it. The recess of the third slot is deeper than those of other slots as shown in FIG. 1-(a), and hence, the resolved row of the third slot is slipped downward as a whole and locational deviation (.DELTA.y) thereof from other slots is produced as shown in FIG. 1-(b). Such relative locational deviation of the rows from each other is called offset distortion.
It is highly demanded to automatically determine the base sequence of the nucleic acids with high accuracy by subjecting digital signals corresponding to the autoradiograph to efficient signal processing, even when such distortion is caused.